Differentiation and Development of the Bovine Blastocyst
Abstract
Differentiation of the blastocyst into epiblast, hypoblast and trophectoderm (TE) are key to establishment of the cell lineages that form fetal, extraembryonic endoderm and placental tissues. The aims were to use the cow as a model to develop markers for specific cell lineages in the blastocyst, understand the role of key molecules in blastocyst differentiation, and gain understanding of the spatial orientation of the processes for formation of the hypoblast and hatching from the zona pellucida. Using RT-PCR of single blastomeres, gene markers were identified for epiblast (AJAP1, DNMT3A, FGF4, H2AFZ, KDM2B, NANOG, POU5F1, SAV1 and SLIT2), hypoblast (ALPL, FGFR2, FN1, GATA6, GJA1, HDAC1, MBNL3, PDGFRA and SOX17) and TE (ACTA2, CDX2, CYP11A1, GATA2, GATA3, IFNT, KRT8, RAC1 and SFN). Moreover, TE contained four subpopulations varying in expression of multiple genes including the TE markers IFNT and EOMES, suggesting that cells were at different degrees of differentiation. In other experiments, it was found that blastocyst differentiation was regulated by two proteins involved in Hippo signaling, YAP1 and AMOT, as well as by the MAPK signaling pathway. Knockdown of either YAP1 or AMOT disrupted blastocyst function as determined by number of TE cells, hatching from the zona pellucida and gene expression. Moreover, knockdown of YAP1 or AMOT affected either number of epiblast or hypoblast cells or expression of genes characteristic of these cells. Addition of a MAPK inhibitor increased the number of epiblast cells without affecting numbers of hypoblast cells. In other experiments, the chemokine CCL24 was identified as being important for spatial organization of the hypoblast since addition of either receptor antagonists or knockdown of CCL24 mRNA decreased the percent of GATA6 + cells (i.e., hypoblast) that were located on the outer portion of the inner cell mass. Finally, it was shown that the blastocyst has no preference for zona hatching through the embryonic or the abembryonic pole. Overall, new markers for studying blastocyst differentiation were identified, heterogeneity of the TE was established and experimental evidence was obtained to show that members of the Hippo signaling pathway, MAPK and selected chemokines play a role in regulating differentiation of the bovine blastocyst.
- Publication:
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Ph.D. Thesis
- Pub Date:
- 2017
- Bibcode:
- 2017PhDT........90N
- Keywords:
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- Animal sciences;Plasma physics;Cellular biology